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MRC LettersReceived: 10 November 2010 Revised: 06 January 2011 Accepted: 11 January 2011 Published online in Wiley Online Library: 29 March 2011

(wileyonlinelibrary.com) DOI 10.1002/mrc.2730

Guaiane-type sesquiterpenoid glucosides fromGardenia jasminoides EllisYang Yu,a,b† Hao Gao,a,c† Yi Dai,a,c Gao-Keng Xiao,a Hua-Jie Zhud andXin-Sheng Yaoa,b∗

Two new guaiane-type sesquiterpenoid glucosides (1 and 2) were isolated from the fruit of Gardenia jasminoides Ellis. Theirstructures were elucidated to be (1R,7R,10S)-11-O-β-D-glucopyranosyl-4-guaien-3-one (1) and (1R,7R,10S)-7-hydroxy-11-O-β-D-glucopyranosyl-4-guaien-3-one (2) by one- and two-dimensional NMR techniques (1H NMR, 13C NMR, HSQC, HMBC and NOESY),MS, CD spectrometry and chemical methods. Copyright c© 2011 John Wiley & Sons, Ltd.

Keywords: 1H NMR; 13C NMR; 2D NMR; CD; Rubiaceae; Gardenia jasminoides; Guaiane-type sesquiterpenoid

Introduction

The genus Gardenia (Rubiaceae) contains more than 250 speciesspread among the warm and tropical regions of the world. Fivespecies are listed in Flora of China and are prescribed in traditionalChinese medicine as sedative, antipyretic, diuretic, cholagogicand anti-inflammatory drugs.[1] G. jasminoides, local Chinese name‘Zhi-zi’, is one of the most popular Gardenia plants and also hasbeen widely used as a yellow dye for staining foods and fabrics.[2,3]

A number of iridoid glucosides, crocin, flavones and quinic acidderivatives have been isolated from the fruit of G. jasminoides andreported as active components.[4 – 7] Our precious investigationsearching active constituents for anti-Alzheimer’s disease led tothe isolation of a series of iridoids glucosides and monoterpenoidglucosides.[8,9] The continuous study of this plant revealed twonew guaiane-type sesquiterpenoid glucosides, which were rareamong the genus Gardenia. The extensive application of one-dimensional (1D) NMR (1H and 13C NMR) and two-dimensional (2D)NMR (COSY, HSQC, HMBC and NOESY) techniques resulted in thestructure elucidation and the 1H and 13C resonance assignmentsof the two glucosides. Furthermore, the detailed conformationanalysis for the seven-membered ring was discussed based onthe NOESY experiments and the coupling constants. The absoluteconfigurations of 1 and 2 were assigned by comparing the resultsof chemical CD calculations and experimental CD spectra.

Results and Discussion

Compound 1 was obtained as a yellow gum, positive to theMolisch reaction. The HR-ESI-Q-TOF-MS gave [M + Na]+ ion atm/z 421.2220, corresponding to the molecular formula C21H34O7,with five degrees of unsaturation. Acid hydrolysis of 1 with 12%hydrochloric acid (HCl) furnished D-glucose, which was identifiedby gas chromatography analysis of the trimethylsilyl imidazolederivative.[10 – 12] The 1H and 13C NMR spectroscopic signals of theglucosyl unit were assigned based on the 1H–1H COSY and HSQCexperiments. The remaining 15 carbon signals, belonging to fourmethyls, four methylenes, three methines and four quaternarycarbons, suggested a skeleton of sesquiterpenoids. A proton

spin system, H-6/H-7/H-8/H-9/H-10(H-14)/H-1, which was deducedfrom the 1H–1H COSY correlations, permitted us to establish thepartial structure of cycloheptane. The unit of α,β-unsaturatedcyclopentanone was elucidated by COSY correlations from H-1 toH2-2 and HMBC cross-peaks at CH3-15/C-3, C-4, C-5 and H-2/C-1, C-3, C-5. Briefly, a carbon skeleton of guaiane-type sesquiterpenoidswas determined by key HMBC correlations at H-6/C-1, 4, 5 andCH3-12, 13/C-7, 11 (Fig. 1). The location of the glucose residuewas established to be at C-11 according to the HMBC cross-peakbetween the anomeric proton H-1′ (δ 4.51) and C-11 (δ 81.0). Fromall these observations, the gross structure of 1 was attributed to asshown in Fig. 2. With the constitution defined, a brief discussion ofthe 13C NMR data is warranted. The low-field chemical shift of theolefinic carbon (C-5 at δ 182.5) was distinctive. It is known that sp2

carbon atoms of alkenes substituted only by alkyl group absorb inthe range of about 100–155 ppm. For 1, a π electron-acceptingsubstituent (carbonyl group) attached to the double bond inducesa α-position deshielding (C-5), which was also attributed to theposition of an exocylic bond of the seven-membered ring. Thus,the distinct chemical shift of C-5 was reasonable and characteristicin guaiane-type sesquiterpenoids.

The CD spectrum showed a positive Cotton effect at 313.0 nmand a negative Cotton effect at 232.8 nm (Fig. 3), suggesting that C-

∗ Correspondence to: Xin-Sheng Yao, College of Pharmacy, Institute of TraditionalChinese Medicine & Natural Products, Jinan University, Guangzhou 510632, P.R. China. E-mail: tyaoxs@gmail.com

† Yang Yu and Hao Gao contributed equally to this work.

a College of Pharmacy, Institute of Traditional Chinese Medicine & NaturalProducts, Jinan University, Guangzhou 510632, P. R. China

b Traditional Chinese Medicine College, Shenyang Pharmaceutical University,Shenyang 110016, P. R. China

c Guangdong Province Key Laboratory of Pharmacodynamic Constituents ofTCM and New Drugs Research, Jinan University, Guangzhou 510632, P. R. China

d Organic Synthesis and Natural Product Laboratory, State Key Laboratory ofPhytochemistry and Plant Resources in West China, Kunming Institute of Botany,Chinese Academic of Sciences, Kunming 650204, P. R. China

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Guaiane-type sesquiterpenoid glucosides from G. jasminoides

Figure 1. Key HMBC (→) and COSY ( ) correlations of 1 and 2.

Figure 2. The structures of compounds 1 and 2.

1 has R configuration and H-1 was located as β orientation.[13,14] InNOESY spectrum, the significant correlations that were observed atH-1/CH3-14 indicatedβ configuration for CH3-14. Proton signal at δ2.12 (1H, t, J = 11.5 Hz) for H-6b showed triplet, implying that therewere a homoallylic coupling for H-6b/H-6a and a vicinal couplingfor H-6b/H-7. The same coupling constant (J = 11.5 Hz) for H-6b/H-7 and H-6b/H-6a suggested H-6b were oriented β-quasi-axial,H-7 were oriented α-quasi-axial. From the above analyses, 1appears more stable in conformation with the subsistent of C-7equatorially oriented and cycloheptane ring in slightly distortedchair conformation, which was found in most sesquiterpenoidswith a known stereochemistry[15,16] and confirmed by our NOESYexperiment (Fig. 4).

In this study, the absolute configuration of C-1was assigned asR by comparing the CD data with the reported. To further confirmthe deduction, CD computation for model compound (R = Me in1) was performed at the B3LYP/6-311++G(2d,p) level.[17,18] Theshapes of the computed CD and experimental CD are almostthe same (Fig. 3). The different �ε after 300 nm between theobserved and the computed may be that the R was methyl usedin the model computations instead of the sugar residue. As thesize of Me is smaller than sugar residue, this may affect thegeometry that had the lowest energy. The difference betweenthe two geometries (R = Me and sugar residue) may lead tothe CD differences between the recorded and the experimental

Figure 4. Key NOESY () correlations of 1.

one. However, the major �ε values near 240 nm are almost thesame between the experimental and computational results. Thisexhibited the absolute configuration for 1 is the same as thepredicted as above. Based on the results, 1 was elucidated as(1R,7R,10S)-11-O-β-D-glucopyranosyl-4-guaien-3-one.

Compound 2 was isolated as a yellow gum, which gave positiveresults to Molisch reaction. Its positive HR-ESI-Q-TOF-MS showed apseudomolecular ion [M + Na]+ at m/z 437.2123 (calcd 437.2151)compatible with molecular formula C21H34O8 (Mw = 414). The1H and 13C spectra of 2 were very similar to those of 1, exceptthat H-7 in 1 was replaced by hydroxy substituent in 2. This wasfurther confirmed by the high-frequency chemical shift of C-7(δ 78.2) and the molecular formula evidence. The structure of 2was assigned by detailed elucidation of 1H–1H COSY, HSQC andHMBC spectra (Fig. 1). The relative and absolutely configuration of2 was determined as the same as 1 by the analysis of the NOESYand CD spectroscopic data (Fig. 3). Thus, 2 was elucidated as(1R,7R,10S)-7-hydroxy-11-O-β-D-glucopyranosyl-4-guaien-3-one.

Experimental

General procedure

Optical rotations were determined on a JASCO P-1020 digitalpolarimeter in CH3OH. IR spectra were measured on a JASCOFT/IR-480 plus spectrometer. UV spectra were recorded in CH3OHusing a JASCO V-550 UV/Vis spectrometer. ESI-MS and HR-ESI-Q-TOF-MS spectra were obtained on a FINIGAN LCQ Advantage MAXmass spectrometer and Micromass Q-TOF mass spectrometer,respectively. The analytical HPLC was performed on a Dionexsystem equipped with a Dionex PDA-100 diode-array detectorusing a RP-18 column (5 µm, 4.6 mm × 250 mm; PurospherSTAR). The preparative HPLC was carried on a Varian instrumentequipped with UV detector (VARIAN Prostar 325, USA) and a RP-18 column (5 µm, 20 mm × 250 mm; Purospher STAR). Columnchromatography was carried on macroporous adsorptive resinsD101 (250–300 µm; Tianjin Pesticide Factory, China), silica gel(200–300 mesh; Qingdao Haiyang Chemical Group Corporation,

Figure 3. The CD spectrum of compounds 1–2 and computed CD curve.

Magn. Reson. Chem. 2011, 49, 258–261 Copyright c© 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/mrc

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Table 1. NMR spectroscopic data (400 MHz for 1H and 100 MHz for 13C) for 1 and 2

1 2

Position δH (ppm), J (Hz) δC (ppm) δH (ppm), J (Hz) δC (ppm)

1 3.04 br. s 48.4 3.02 br. s 48.5

2 2.35 dd (18.3, 6.0); 2.05 dd (18.1, 3.5) 38.3 2.32 m 38.6

3 212.2 213.4

4 137.4 142.3

5 182.5 175.4

6 3.26 m; 2.12 t (11.5) 30.7 3.08 m; 2.52 d (12.6) 36.5

7 1.47 m 53.0 78.2

8 1.99 br. m; 1.34 m 32.1 2.35 dd (13.2, 5.9); 1.80 br. d (12.1) 30.9

9 1.42 m; 1.09 m 31.0 1.76 m; 1.23 m 27.3

10 2.21 m 35.0 2.15 m 35.8

11 81.0 89.2

12 1.30 s 25.6 1.40 s 25.7

13 1.27 s 22.2 1.26 s 25.6

14 0.98 d (7.1) 20.4 0.96 d (7.0) 19.8

15 1.71 d (2.1) 7.8 1.70 d (2.0) 9.8

Glc-1′ 4.51 d (7.7) 98.6 4.58 d (7.6) 98.0

2′ 3.18 m 75.5 3.19 m 76.4

3′ 3.36 m 78.6 3.09 m 77.7

4′ 3.26 m 71.9 3.16 m 71.9

5′ 3.24 m 77.6 3.29 m 78.8

6′ 3.83 dd (12.1, 2.0); 3.63 dd (11.8, 5.4) 63.0 3.74 dd (11.5, 2.2); 3.46 dd (11.6, 5.7) 63.3

Qingdao, China), Sephadex LH-20 (Amersham Biosciences AB),Toyopearl HW-40 (Toyo Soda MFG) and ODS (60–80 µm; Merck).TLC was performed on silica gel GF254 plates (Qingdao HaiyangChemical Group Corporation).

NMR spectra

NMR spectra were recorded on a Bruker AVANCE 400 NMRspectrometer equipped with a 5-mm BBO z-gradient probe head.1H, 13C NMR spectra (400 and 100 MHz, respectively) and 2DNMR experiments (COSY, HSQC, HMBC, NOESY) were recorded at300 K using standard Bruker pulse programs (XWin-NMR version3.5).

About 3.0–10.0 mg samples were dissolved in 0.6 ml deuteratedsolvents (CD3OD), which was used as the internal lock. Thechemical shifts were given in δ (ppm) scale and referenced tothe solvent signal (δ (CHD2OD) = 3.31 ppm for 1H NMR and δ

(CD3OD) = 49.0 ppm for 13C NMR).For 1H NMR, 8–16 transients were acquired with a 2.0-s

relaxation delay, 32K data points, 3205 and 4006 Hz spectral widthfor 1 and 2 and 90◦ pulses (14.00 µs at 0 dB). FIDs were Fouriertransformed with LB = 0.1 Hz and the spectra were zerofilled to32K points. For 13C NMR, 3000–10 000 transients requiring 2–8 hacquisition time were acquired with 2.0-s relaxation delay and24 149 Hz spectral width and the 90◦ pulse (7.5 µs at −4 dB).Fourier transformed with LB = 1.0 Hz and the spectra werezerofilled to 32K points. The resulting spectra were manuallyphased, baseline corrected, calibrated and integrated using Xwin-NMR software.

1H–1H magnitude-mode ge-2D COSY spectrum was recordedover 1K data points in F2 and 256 data points in F1, using a 2.0-srelaxation delay and 4000 Hz spectral width in both dimensions.Window function for COSY spectra was Qsine (SSB = 0). Thephase-sensitive ge-2D HSQC spectrum was recorded over 1K data

points in F2 and 256 data points in F1, using a 2.5-s relaxation delay,2394 Hz spectral width in F2 and 14 087 Hz in F1. Magnitude-modege-2D HMBC spectrum was recorded over 2K data points in F2 and256 data points in F1, using a 2.0-s relaxation delay and 4000 Hzspectral width in F2 and 24 149 Hz in F1. The optimized couplingconstants for HSQC and HMBC spectra were 1J(C, H) = 140 Hzand nJ(C, H) = 10 Hz, respectively. Qsine (SSB = 2) was usedfor the window function of HSQC and HMBC processing. Thephase-sensitive NOESY experiment was obtained using a mixingtime of 300 ms, a 2.0-s relaxation delay and the 4000 Hz spectralwidth for both dimensions. The spectra were zerofilled to 1Kdata points, and Qsine (SSB = 3) was used for the windowfunction.

Plant material

The fruit of G. jasminoides was collected from Guangzhou QingpingMedical Material Market, China, in April 2007 and authenticated byProfessor Danyan Zhang, Guangzhou Chinese Medicine University.A voucher specimen (20 070 417) is deposited in the Institute ofTraditional Chinese Medicine & Natural Products, Jinan University,Guangzhou, China.

Extraction and isolation

Dried fruit of G. jasmonoides (8.0 kg) was refluxed with 60% (v/v)EtOH for three times. After evaporation of EtOH in vacuo, theaqueous residue was subjected to column chromatography overD101 eluted with EtOH/H2O to yield five fractions. Fraction 3(EtOH/H2O, 50 : 50 v/v) was separated by silica gel eluted withCHCl3/MeOH gradiently to yield subfractions 3–7 (CHCl3/MeOH,9 : 1 v/v), which was submitted to repeated ODS, Toyopearl HW-40 columns eluted with MeOH/H2O, followed by preparativeHPLC (MeOH/H2O, 4 : 6 v/v) to yield compounds 1 (3.0 mg) and2 (15.7 mg).

wileyonlinelibrary.com/journal/mrc Copyright c© 2011 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2011, 49, 258–261

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Guaiane-type sesquiterpenoid glucosides from G. jasminoides

(1R,7R,10S)-11-O-β-D-glucopyranosyl-4-guaien-3-one (1)

Yellow gum; [α]25.6D − 21.2 (c 0.5, MeOH); UVλmax (MeOH) nm (log

ε): 244 (4.17); IR (KBr) νmax (cm−1) 3415, 2925, 1679, 1632, 1456,1382, 1078; CD (c = 0.025, MeOH): 232.8 nm (�ε − 3.40), 313 nm(�ε+1.32); 1H (400 MHz) and 13C NMR (100 MHz) (Table 1); HR-ESI-Q-TOF-MS m/z 421.2220 [M + Na]+ (calculated for C21H34O7Na,421.2202).

(1R,7R,10S)-7-hydroxy-11-O-β-D-glucopyranosyl-4-guaien-3-one (2)

Yellow gum; [α]23.2D −15.8 (c 0.5, MeOH); UVλmax (MeOH) nm (log ε):

244 (4.24); IR (KBr) νmax (cm−1): 3441, 2926, 1644, 1454, 1393, 1038;CD (c = 0.025, MeOH): 235 nm (�ε − 3.00), 318.4 nm (�ε + 1.36);1H (400 MHz) and 13C NMR (100 MHz) (Table 1); HR-ESI-Q-TOF-MSm/z 437.2123 [M + Na]+ (calculated for C21H34O8Na, 437.2151).

Computational

The effect of the sugar moiety on ECD should be weak due to itsflexibility and the lack of double bond near the stereogenic centers.ECD was computed using reasonable simplified model which is touse OMe to replace the sugar residue. Conformational search wasperformed using Amber force field and the conformations withlow energy from 0 to 3 kcal/mol were optimized at the B3LYP/3-21G(d) level. Optimization for the selected lowest geometry wasthen performed at the B3LYP/6-311+G(d,p) level. This geometrywas then used in ECD computations at the B3LYP/6-311++G(2d,p)level.

Acknowledgements

This work was supported by grants from the National NaturalScience Foundation of P. R. China (No. 30701047) and the KeyProject of the Chinese Ministry of Education (No. 208173).

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Magn. Reson. Chem. 2011, 49, 258–261 Copyright c© 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/mrc